Although the mouse is the pre-eminent model to study gene function in development, the process of targeted gene deletion using homologous recombination in ES cells can be inefficient, costly and slow. In addition, when there are multiple genes with overlapping, compensatory roles in development, all must be targeted to tease out function. The recent application of RNA interference (RNAi) to ES cells and recently to embryos provides the opportunity to target multiple genes, and when combined with a tissue specific promoter can specify the timing or tissue of knock-down. Despite the potential, surprisingly little is understood regarding the uptake of RNAi constructs into individual cells and tissues, and a variety of delivery methods from naked DNA, lipoprotein delivery, viral delivery, to electroporation have been employed with varying success. We have delivered constituitively expressed small hairpin (sh) RNAs to pregnant mice during the early postimplantation development and observed gene knock-down and developmental defects that phenocopy the null embryo. In the current investigation, we propose a careful, systematic evaluation of the factors that influence successful gene targeting using this approach. These studies will be followed by an assessment of the ability of a tissue specific promoter to drive RNAi in early embryos. These nested "proof of concept" studies are required to understand and control the variables involved in a potentially powerful approach to gene silencing in the embryo. To study gene function in development, scientists "target" or remove genes in mouse embryos. The process is both complicated and expensive, so we have developed a new, rapid method to "knock down" genes in the mouse embryo. We propose a series of studies to determine the reliability and efficiency of these techniques. This approach should provide new data on the role of individual genes, and in the long term may have important implications for correcting genetic defects in the fetus. [unreadable] [unreadable] [unreadable]